SYNAPTIC AVAILABILITY OF GLUN2A SUBUNIT OF NMDA RECEPTORS FROM PHYSIOLOGICAL MECHANISMS TO PATHOLOGY: THE ROLE OF RABPHILIN 3A

Background and Purpose - NMDA receptor subunit composition strictly commands receptor function and pharmacological responses. The identity of the GluN2 subunit regulates biophysical and pharmacological properties of the receptor and influences receptor assembly, signaling and trafficking to the postsynaptic membrane. The number or subunit composition of NMDA receptors vary during activity dependent plasticity and CNS disorders like Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID) where you can find an enhanced extrasynaptic localization of GluN2B-containing NMDA receptors and an increase of the GluN2A/GluN2B ratio at striatal synapses. Recently, a two-hybrid screening to find potential proteins interacting with the C-terminal tail of the GluN2A subunit of the NMDA receptor has highlighted Rabphilin 3A (Rph3A) as a potential partner. Rph3A is a synaptic vesicle-associated protein that was first identified as a binding partner of the GTP-bound Rab3A, a member of the Rab family of GTPases implicated in vesicle docking/fusion reactions. Moreover, different studies have indicated that Rph3A can regulate exo- and endocytosis processes at synaptic sites. Our goal was to characterize Rph3A interaction with GluN2A at postynaptic sites and to assess its relevance in PD and LID. Methods and Results - Firstly, we verified its interaction with GluN2A by immunoprecipitation and GST pull-down experiments. Subcellular fractionation assays revealed that Rph3A is present in Triton Insoluble Postsynaptic compartment and post-synaptic density (PSD) fractions. Immunofluorescence studies performed in neuronal hippocampal cultures, confirmed biochemical experiments revealing Rph3A colocalization with PSD-95 (marker of the postsynaptic compartment) and GluN2A. Moreover, we found Rph3A to be interacting with a 40AA domain on C-terminal tail of GluN2A between AA 1349 and 1389. Therefore, we designed a cell permeable peptide containing this sequence called TAT-2A-40. This peptide is capable of disrupting the interaction between Rph3A and GluN2A. By doing this, it can reduce the expression of GluN2A in dendritic spines and its surface expression as we show by immunocytochemistry and electrophysiology. Moreover, we have observed an increase of GluN2A/Rph3A interaction in a rat PD model as well as a rat LID model. We have that the TAT-2A-40 was capable of significantly reducing the dyskinetic behavior in these animals. Conclusions - These results strongly indicate the presence of Rph3A in the PSD compartment and suggest a function of Rph3A in the modulation of NR2A localization. Rph3A could play a crucial role in the stabilization of GluN2A subunits at the plasma membrane in dendritic spines. Therefore, this interaction could be a potential therapeutic target for diseases where NMDAR composition is significantly altered such as Parkinson's Disease and L-DOPA-induced dyskinesia.